Detection of femtomole quantities of mature cathepsin K with zymography - PubMed (original) (raw)
. 2010 Jun 1;401(1):91-8.
doi: 10.1016/j.ab.2010.02.035. Epub 2010 Mar 3.
Affiliations
- PMID: 20206119
- DOI: 10.1016/j.ab.2010.02.035
Detection of femtomole quantities of mature cathepsin K with zymography
Weiwei A Li et al. Anal Biochem. 2010.
Abstract
Cathepsin K, the most potent mammalian collagenase, has been implicated in osteoporosis, cancer metastasis, atherosclerosis, and arthritis. Although procathepsin K is stable and readily detected, the active mature cathepsin K eludes detection by in vitro methods due to its shorter half-life and inactivation at neutral pH. We describe, for the first time, reliable detection, visualization, and quantification of mature cathepsin K to femtomole resolution using gelatin zymography. The specificity of the method was validated with cathepsin K knockdown using small interfering RNA (siRNA) transfection of human monocyte-derived macrophages, and enzymatic activity confirmed with benzyloxycarbonyl-glycine-proline-arginine-7-amino-4-methylcoumarin (Z-GPR-AMC) substrate hydrolysis was fit to a computational model of enzyme kinetics. Furthermore, cathepsin K zymography was used to show that murine osteoclasts secrete more cathepsin K than is stored intracellularly, and this was opposite to the behavior of the macrophages from which they were differentiated. In summary, this inexpensive, species-independent, antibody-free protocol describes a sensitive method with broad potential to elucidate previously undetectable cathepsin K activity.
Copyright 2010 Elsevier Inc. All rights reserved.
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